For several years we have studied replication initiation in specific DNA sequences. Our development of Single Molecule Analysis of Replicated DNA (SMARD) has allowed us to study the replication of segments encompassing up to 1 Mb of a genomic locus. In the present proposal we will use this powerful approach to examine replication in three model systems: the Igh locus, human and mouse telomeres, and the triplet repeat- containing Fragile X Mental Retardation (FMR1) locus. We have shown that origins are absent in a region of the IgH-constant region locus in non-B cells but are activated at preferred sites in pro-B cells. We will use Igh- C locus primarily as a model to examine responses to replication stress, including activation of latent origins. We will determine pre-replication complex (pre-RC) localization and nucleosome organization in the Igh-C locus by ChIP. Comparison of SMARD origin mapping with pre-RC occupancy and nucleosome landscape will provide insights into the mechanisms of latent origin activation. In the second model we have made essential technical advances in SMARD that allow us to study replication in specific telomere loci and have obtained detailed information on telomere replication programs. A key finding is that human and mouse telomeres appear to be replicated by chromosome-specific replication programs rather than a universal program. We will use SMARD to determine how these programs are affected by replication stress and what aspects of replication are challenged by telomeres. We will also examine telomere dysfunction at specific telomeres. These analyses will reveal the flexibility of telomere replication programs, including the ability to activate latent origins, and establish if specific replication programs render telomeres more susceptible to stress and dysfunction. We also have evidence to suggest that BLM helicase directly contributes to efficient telomere replication. We will use SMARD to determine how BLM facilitates telomere replication and if telomeres are more reliant on BLM than internal genomic regions. We will also determine the functional overlap between BLM and other helicases in telomere replication. In the third model we will study trinucleotide repeat (TNR) expansion, which is associated with more than 30 inherited diseases including Fragile X syndrome (FXS). Many model systems have been generated to study the role of replication in TNR expansion, but models of expansion at endogenous human loci are lacking. Using SMARD we are now able to compare the role of replication initiation sites in TNR expansion in the endogenous FMR1 locus (the affected locus in FXS) in human cells, including cell lines from women at risk for giving birth to FXS males, that have and do not have TNR expansion to elucidate the role of origin site selection on TNR instability. We will also determine the contribution of CTCF binding to FMR1 replication programs. Based on our preliminary data, we expect these proposed studies will both greatly increase our understanding of telomere and TNR replication and allow us to establish new paradigms for the role replication origin selection in health and disease.